Deoxyribonucleoside triphosphate pools in Neurospora crassa: effects of histidine and hydroxyurea

An effective HPLC method for detecting deoxyribonucleoside triphosphates in hyphae from the fungus Neurospora crassa has been developed. In rapidly growing cells the nucleotide levels vary from 11.8 pmoles/micrograms DNA for dGTP to 24.2 pmoles/micrograms DNA for dTTP. These levels fall by approximately one half in stationary-phase cultures but the ratio of each pool to dGTP remains the same. The dNTP pools in conidia are at least 5-fold lower than in rapidly growing cells. The pool sizes are the same in static and shaking cultures. When the ribonucleotide reductase inhibitor, hydroxyurea (30 mM), is added to rapidly growing cultures, DNA synthesis is stopped and the dGTP pool is reduced by 39%, while the size of the other pools remains the same. In the presence of 11 mM histidine, DNA synthesis is also stopped and the size of the dGTP pool reduced by 46% while the deoxypyrimidine pools are somewhat increased. This suggests that the toxicity of excess histidine in Neurospora may be due to its ability to interact with the ribonucleotide reductase, inactivating the enzyme. Histidine may react with the free radical at the active site, as does hydroxyurea.